Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells


  • Su Mi Choi,

    1. Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD
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  • Yonghak Kim,

    1. Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD
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  • Joong Sup Shim,

    1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD
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  • Joon Tae Park,

    1. Department of Surgery and the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD
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  • Rui-Hong Wang,

    1. Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
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  • Steven D. Leach,

    1. Department of Surgery and the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD
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  • Jun O. Liu,

    1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD
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  • Chuxia Deng,

    1. Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
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  • Zhaohui Ye,

    Corresponding author
    1. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD
    • Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Yoon-Young Jang

    Corresponding author
    1. Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD
    2. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD
    • Stem Cell Biology Program, Department of Oncology, Johns Hopkins University School of Medicine, 1550 Orleans Street, CRB2 Room 552, Baltimore, MD 21231
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    • fax: 410-502-5742

  • Potential conflict of interest: Nothing to report.

  • This work was supported by the National Institutes of Health (NIH; grant no.: AA020020) and the Maryland Stem Cell Research Fund (MSCRF; grant nos.: 2010-MSCRFII-0101-00, 2011-MSCRFE-0087, and 2012-MSCRFF-0152-00). This work was also supported by the NIH Center for Regenerative Medicine. S.D.L. is supported by the NIH (grant no.: DK61215) and the Paul K. Neumann Professorship at Johns Hopkins University. The maintenance of Johns Hopkins Drug Library was supported, in part, by the National Cancer Institute (R01CA122814), the Flight Attendant Medical Research Institute, and by grant number UL1 RR 025005 from the National Center for Research Resources, a component of the NIH and the NIH Roadmap for Medical Research (to Johns Hopkins School of Medicine).


Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease technology, we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications. Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. (HEPATOLOGY 2013;57:2458–2468)

Some of the biggest challenges modern medicine faces are the long timeline (>12 years), high failure rate (∼95%), and cost (>$1 billion) associated with developing a single new drug.1, 2 The development of novel compounds has been accelerating as a result of the genome-driven discovery of new drug targets, expansion of natural and synthetic chemistry compound collections, and development of high-throughput screening technologies.3, 4 Despite these advances, frequent attrition of a lead series occurs as a result of unfavorable drug absorption, distribution, metabolism, excretion, and/or toxicity (ADMET),1, 2, 5 indicating a lack of sufficient predictability of traditional drug-screening tools, such as cancer cell lines and animal models. To avoid such high failure rate in late stages of the drug-developmental process, more patient-relevant screening platforms need to be developed for early-stage drug screens.

The emergence of patient-specific iPSC technology and disease models established from these cells, which may provide renewable sources for a highly patient-relevant and powerful throughput screening platform, has brought high enthusiasm in the field; not only could a patient's iPSCs be used to generate cells for transplantation to repair damaged tissues, but also the differentiated progeny of such cells could be used to recapitulate disease phenotypes and enable more-efficient drug screening to find new treatment of the disease.6-14 To realize such potential of iPSCs, we and others have generated patient-specific iPSCs from various human tissues and differentiated these cells into different somatic cell types, including blood and liver cells, in the past few years.6-8, 10-13 More recently, we and others have demonstrated that iPSCs derived from patients with multiple metabolic liver diseases, including alpha-1 antitrypsin (AAT) deficiency, could indeed be utilized for disease modeling after differentiation into hepatocyte-like cells.6, 7, 15, 16 However, it remains elusive whether these cellular models of liver diseases can be effective for drug screening and discovery.

AAT deficiency is one of the common genetic disorders of the liver.17 Importantly, AAT deficiency can progress to severe liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC).17-19 Currently, there is no drug or gene therapy available to treat liver disease or prevent its progression into cirrhosis and HCC. The most common clinical form of AAT deficiency is associated with the PiZ variant of this protein, which is caused by a (G>A) point mutation at codon 342 (Glu342Lys) in exon 5 of the AAT gene.19 The mutation promotes spontaneous polymerization and retention of the polymers in the endoplasmic reticulum (ER) of hepatocytes, resulting in protein overload that, in turn, causes the liver diseases.18 The deficiency of AAT in plasma predisposes the affected individuals to chronic pulmonary diseases as well. Augmentation therapy has been given for treatment of lung disease, but there is no therapy available other than liver transplantation for individuals with AAT-deficiency–related liver disease. Because it is unlikely that current AAT augmentation therapy will alter the course of AAT liver disease and the organ supply for transplantation is limited, alternative therapeutic strategies are required to prevent or treat both liver and lung failure by tackling the cause rather than the symptoms. The potential of human iPSC-based therapy is therefore very attractive for diseases such as AAT deficiency; therefore, it will be of value to develop therapeutic strategies to (1) pharmacologically decrease the mutant AAT accumulation or (2) correct the disease-causing mutation for gene- and cell-based therapy. Using our established iPSCs derived from AAT-deficiency patients,6, 7 we explored the feasibility of developing both therapeutic strategies.

To expedite the eventual application of lead compounds to patients, we have employed our established clinical ready drug library (the Johns Hopkins Drug Library; JHDL)20 for iPSC-based drug screening. The JHDL currently consists of 3,131 clinical drugs (including 2,800 drugs that either have been approved by U.S. Food and Drug Administration [FDA]/foreign counterparts or have entered phase II clinical trials).21 Therefore, any hit from this library can bypass early stages of preclinical and clinical studies before clinical testing in parents. Although a few reports, including our own, have shown the feasibility of testing several candidate drugs with iPSC-based models,7, 22, 23 there have been no reports of large-scale drug screening in a blind manner using a patient iPSC-based disease model. To our knowledge, this is the first report of a large-scale drug screening using an iPSC-based disease model.

To develop potential gene and cell therapy, there have also been efforts to enhance the low efficiency of site-specific gene correction in human iPSCs, including the demonstration of zinc finger nuclease (ZFN)-mediated gene targeting for various genes, including the high-efficiency correction of the AAT gene.24-29 Although the application of ZFNs represents a significant improvement over the traditional targeting technologies, the design of ZFNs has been a formidable engineering challenge, preventing its broad applications in research laboratories. Therefore, we assessed the efficacy of the recently developed transcription activator-like effector nuclease (TALEN) technology30-34 for targeted gene correction of liver disease mutation in patient-specific iPSCs. Here, we report on the application of patient-specific iPSCs in drug screening (and the discovery of new uses of already approved clinical drugs) as well as for highly efficient gene targeting.


AAT, alpha-1 antitrypsin; ADMET, absorption, distribution, metabolism, excretion and/or toxicity; ALB, albumin; CBZ, carbamazepine; CK18, cytokeratin 18; CYP, cytochrome P450; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FDA, U.S. Food and Drug Administration; Gli, glipizide; GSK-3β, glycogen synthase kinase 3 beta; HCC, hepatocellular carcinoma; HD, Huntington's disease; HDAC, histone deacetylase; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; JHDL, Johns Hopkins Drug Library; Li, lithium; MH, mature hepatocyte; mTOR, mammalian target of rapamycin; PAS, periodic acid-Schiff; PASD, PAS with diastase digestion; PCR, polymerase chain reaction; TALEN, transcription activator-like effector nuclease; Thi, thiamine; VPA, valproic acid; ZFN, zinc finger nuclease.

Materials and Methods

Drug Screen Using a High-Throughput Immunofluorescence Analyzer.

All human iPSCs were cultured on Matrigel (BD, Franklin Lakes, NJ) using mTeSR (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) and differentiated into hepatic cells, as we described previously,6, 7, 10 with some modification (see Supporting Materials for details). This study was done in accord with Johns Hopkins Institutional Stem Cell Research Oversight regulations and following a protocol approved by the Johns Hopkins Institutional Review Board.

An initial screen of all compounds from the JHDL,20 which includes 3,131 clinical compounds, was conducted using one of our AAT deficiency patient iPSC lines (iAAT2), propagated using the differentiation method described above in 96-well imaging plates. iPSC-derived hepatic cells were treated with JHDL compounds (one per well) at a final concentration of 5 μM for 4 days at the end of hepatic maturation. After drug treatment, AAT immunofluorescence (IF) staining was performed and the total AAT fluorescence intensity of each well was measured using a Safire2 microplate reader. Results obtained from this fluorescence-based high-throughput assay were normalized with signals from 4',6-diamidino-2-phenylindole–labeled nuclei. Percentages of changes of AAT signals were calculated by dividing with that of dimethyl-sulfoxide–treated samples.

For confirmatory screening, we carefully selected 43 compounds without major side effects from 262 compounds that inhibited AAT accumulation by >50%. We then further tested these drugs using four different AAT-deficiency patient iPSC lines (iAAT2, iAAT3, iAAT45, and iAAT25) and freshly prepared drugs, rather than using the stock. Experiments were repeated four times with consistent results. Using the same IF assay, we obtained five hits, which consistently show the effect in multiple patient iPSCs.

See Supporting Materials for hepatic differentiation, TALEN-mediated AAT gene-targeting, periodic acid-Schiff (PAS) with diastase digestion (PASD), cytochrome P450 (CYP) assay, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (PCR), and statistics.


Optimization of the iPSC-Based Hepatic Differentiation Protocol for Large-Scale Drug Screening.

The molecular basis of AAT deficiency has been shown to be the accumulation of AAT protein as ordered polymers within the ER of hepatocytes.19, 35 We have previously demonstrated the formation of intracellular globules that are formed by the polymers of mutant AAT proteins within AAT-deficiency iPSC-derived mature hepatocyte-like cells by PASD.7 We also showed that carbamazepine (CBZ), which has been shown to decrease the hepatic load of mutant AAT protein and hepatic fibrosis in a mouse model of AAT-deficiency–associated liver disease,35 could reduce the AAT accumulation in AAT-deficiency patient iPSC-derived hepatocyte-like cells using the PASD assay.7 However, this assay does not permit automated quantification of readout using a high-throughput format IF/luminescence reader; therefore, it is not optimal for a large-scale compound screening. To perform efficient, reliable screening using a high-throughput format IF reader and our iPSC model of AAT-deficiency liver disease,7 we have modified our hepatic differentiation protocol to be compatible with a 96-well format, followed by IF staining with a specific antihuman AAT antibody, permitting visualization and quantitative detection of AAT accumulation within hepatic cells (Fig. 1 and Supporting Fig. 1). To achieve this goal, one replating step was added at the end of the hepatic progenitor stage to evenly distribute iPSC-derived hepatic cells into 96-well plates without losing viability or functionality (Fig. 1A and Supporting Figs. 2 and 3). Both the definitive endoderm cells at an early differentiation stage and the fully mature hepatocyte (MH)-like cells were excluded for this purpose because of their lower viability associated with enzymatic dissociation and the replating step. Of note, no animal feeder cells were used throughout the iPSC expansion and differentiation processes. With future application of the hits in clinics in mind, we purposely selected a relatively low concentration (5 μM), from the reported doses of 5-20 μM employed for other studies using the same drug library, for this screening.36-39 Our selected screening dose is within a physiological concentration of a large number of drugs in this library.36-39 Using CBZ, with which we previously demonstrated the reduction of AAT accumulation in patient iPSC-derived hepatocyte-like cells,7 we determined whether the modified differentiation protocol and selected dosage can reproduce similar results when analyzed by a high-throughput format IF reader. Compared to nonpatient iPSC healthy controls, the patient iPSCs (PiZZ, the most common, severe form of AAT deficiency) consistently generated a higher AAT signal within mature hepatic cells after differentiation (Fig. 1B,C), indicating intracellular AAT accumulation. When treated with CBZ, the hepatocyte-like cells derived from patient iPSCs exhibited significant reduction in the intracellular retention of the AAT proteins, determined by both high-throughput format IF reader and microscopy (Fig. 1B,C). This result was indeed consistent with the PASD result of these iPSC-derived hepatocyte-like cells, further confirming the validity of the IF-based assay (Fig. 1D).

Figure 1.

Optimization of the iPSC-based hepatic differentiation protocol for high-throughput format drug screening. (A) Diagram summarizing our modified hepatic differentiation protocol used for the high-throughput format drug screening. Human iPSCs that had been maintained in a feeder-free condition were differentiated into hepatic progenitor cells before replating into collagen 1–treated 96-well imaging plates, which are specifically designed for a throughput IF reader. (B) Detection of intracellular AAT with a high-throughput format IF reader. Control iPSC (iH71)- or patient iPSC (iAAT2)-derived mature hepatic cells (105/well) in 96-well microplates were treated with CBZ (5 μM) for 4 days. AAT staining was performed on 96-well plates, and AAT fluorescence was obtained using a Safire2 microplate reader. RFU, relative fluorescence unit. (C) IF images of AAT expression obtained from differentiated hepatocyte-like cells of control iPSCs (iH10, left panel) and an AAT-deficiency patient iPSC line (iAAT3, middle panel). Compared to control iPSCs, increased amounts of AAT/AAT globules (red) were observed in iAAT cells after hepatic maturation. Expression level was significantly decreased after CBZ treatment (5 μM for 4 days, right panel). (×100). (D) PASD images of these iPSC-derived hepatocyte-like cells. Numerous PASD+ inclusion bodies (pink) were detected within MH-like cells derived from the AAT patient iPSCs (iAAT3, middle panel), but not in control iPSC (iLC2)-derived hepatocyte-like cells (left panel). Level of PASD+ inclusion body/globules was decreased with CBZ treatment (5 μM for 4 days, iAAT3, right panel). (×100).

Drug Screening Using Patient-Specific iPSCs.

Blind screening of the clinical-ready drug library (JHDL) was initiated with our AAT-deficiency patient iPSC-derived hepatocyte-like cells using the optimized screening assay (Fig. 2).

Figure 2.

Patient-specific iPSC-based drug-screening process and results. Flow chart depicting the drug-screening process and result summary. After the first screening of all JHDL drugs (3,131 clinical compounds) with the IF-based screening assay (Fig. 1), 262 compounds, which significantly decreased the accumulation of AAT to less than 50% of nontreated controls, within hepatocyte-like cells derived from 1 AAT-deficient patient iPSC (iAAT2), were selected for further testing. Of the 262 compounds, we carefully selected 43 drugs, which are relatively safer, without significant unwanted effects to any of the major organs. For the confirmatory screening for the 43 drugs in multiple iPSC lines, including iAAT2, iAAT3, iAAT45, and iAAT25, we used independently prepared drugs, rather than using the JHDL stock. Of the 43 compounds, we discovered five final drug hits, which consistently showed the AAT-reducing effect in the multiple patient-derived hepatocyte-like cells. The rest of the drugs did not repeat the results with either one or multiple iPSC lines. Representative results obtained from multiple independent iPSC lines are shown to demonstrate the AAT reduction effects of the final five drug hits in the confirmatory screening (5 μM each for 4 days). *P < 0.05; **P < 0.01. RFU, relative fluorescence unit.

The initial screen of the entire drug library, at a final concentration of 5 μM of each drug, yielded 263 hits (Supporting Table 1). Drugs that decreased average total fluorescence intensity within the AAT-deficiency patient iPSC-derived hepatocyte-like cells by more than 50% were considered as hits (Fig. 2). A majority of these were antidepressant, -convulsant, and -biotics (Supporting Table 1). Then, we performed an extensive, systematic literature search on the hits (e.g., mechanisms of action, target proteins, pharmacokinetics, and so on) and prioritized the hits based on approved status, main effects, and side effects. Among these 262 compounds, 43 drugs, which are FDA approved or have a history of clinical application internationally and without significant unwanted/side effects to any of the major organs, were chosen for further study (Fig. 2; Supporting Table 2). Drugs that have known cytotoxicity (e.g., anticancer drugs) or predicted side effects related to their intended use (e.g., antihypertension drugs) on patients were excluded from further screening.

To confirm the effects of the selected hits and further identify a more-universal candidate drug(s) that is effective in diverse patients, we used separate preparations of the drugs than the original stock of the JHDL and tested the selected 43 hits on hepatocyte-like cells derived from four different patient-iPSC lines (iAAT2, iAAT3, iAAT45, and iAAT25; Fig. 2 and Supporting Fig. 2). After confirmatory screening steps with these multiple iPSC lines, we were able to discover the final five hits, which consistently showed the similar effects in multiple patient-derived hepatocyte-like cells (Fig. 2; Table 1). Similar effects were observed in the range of 2.5-10.0 μM (Supporting Fig. 4). The rest of the drugs failed to show the effects in one or more iPSC lines (Fig. 2).

Table 1. Characterization of the Final Hits
Drug NameIndicationApproval StatusReported Mechanism of Action
  • Abbreviations: IP3, inositol 1,4,5-tris-phosphate; ATZ, antitrypsin Z; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.

  • *

    These drugs share a common mechanism.

CBZ*AnticonvulsantFDAReduces inositol and IP3 levels; autophagy enhancer; reduces ATZ accumulation in mice41, 47
GliAntidiabeticFDAATP-dependent K+ channel blocker; increases insulin release from pancreatic beta cells
Li*AntipsychoticFDAInhibits IMPase and reduces inositol and IP3 levels41; induces autophagy by inhibiting inositol monophosphatase40, 42 as well as antimanic and hematopoietic activities; interferes with transmembrane sodium exchange in nerve cells by affecting Na+, K+-ATPase; affects cAMP concentrations
VPA*AnticonvulsantFDAAnticonvulsant that also has efficacy as a mood stabilizer in bipolar disorder44; reduces inositol and IP3 levels41
ThiVitaminFDAVitamin B1, antioxidant, erythropoietic, mood modulating, and glucose-regulating activities; reacts with ATP to form an active coenzyme, thiamine pyrophosphate; thiamine pyrophosphate is necessary for the actions of pyruvate dehydrogenase and alpha-ketoglutarate in carbohydrate metabolism and for the actions of transketolase, an enzyme that plays an important role in the pentose phosphate pathway.

Interestingly, one of the hits, after the blind screening, turned out to be CBZ, which strongly validated the functionality of our drug-screening assay. Importantly, three of the five hits were well-known clinical mood-stabilizing drugs sharing a similar mechanism of action (Table 1). Inositol depletion seems to be a common mechanism for the following thee drugs: lithium (Li), CBZ, and valproic acid (VPA).35, 40, 41 Consistent with a role for inositol depletion in autophagy regulation, these three drugs have been known to enhance the clearance of aggregate-prone proteins in several nonhuman conditions associated with either liver or neurodegenerative diseases.19, 40-42

To figure out the mechanisms underlying the observed effects of these drugs, we measured AAT levels in cells and media using ELISA (Supporting Fig. 5) and analyzed the protein expression of an autophagy marker (LC3) and AAT gene expression (Supporting Fig. 6). We found that intracellular AAT protein levels were indeed decreased after drug treatments, whereas the protein level in media and gene expression did not change after treatments (Supporting Figs. 5 and 6). Interestingly, LC3 was increased in cells treated with the five drugs (Supporting Fig. 6), including two drugs (glipizide [Gli] and thiamine [Thi]), which have not been previously reported on as autophagy enhancers (Table 1). These data collectively suggest that the mechanism of the final drug candidates is likely the result of the autophagy-mediated degradation of folded AAT proteins, rather than decreasing synthesis of AAT.

TALEN-Mediated Efficient Gene Correction of AAT Z Mutation in Patient-Specific iPSCs.

Future autologous cell-replacement therapy for AAT deficiency will require the gene correction of the Z mutation in patient cells. To assess the efficacy of TALEN technology in gene targeting of liver disease mutations, we designed and constructed a pair of TALEN expression vectors that specifically recognize the flanking sequences of the Z mutation of the AAT gene (Fig. 3). To compare this approach to the reported ZFN technology, we utilized a previously reported donor construct,24 which also allows the clean excision of integrated DNA from the genomes, in combination with the TALEN vectors (Fig. 3A).

Figure 3.

TALEN-mediated gene correction of AAT mutation in patient-specific iPSCs. (A) Strategy for precise genome modification using TALEN. Genomic DNA sequence around the Z mutation (“A” in red) in the human AAT gene is shown. A pair of TALENs were designed to specifically recognize the sequences flanking the point mutation with a 16-bp spacer. The targeting vector (homologous recombination [HR] donor)24 contains the wild-type sequence (“G” in green) in the homology arm. Selection marker expression cassette PGK-puroΔtk is flanked by piggyBac-inverted terminal repeat sequences, which allow precise excision upon transient expression of piggyBac transposase. Note that, to facilitate this transpose-mediated seamless DNA excision, the endogenous sequence, CTGA, was changed to TTAA without alteration of the amino acid sequence. Confirmation of gene-targeting events by PCR (B) and southern blotting (C). (B) Genomic DNAs isolated from the picked puromycin-resistant clones after transfection of TALENs and targeting vector were screened by PCR. A 1.2-kb PCR product indicates the targeted events. Using a separate pair of primers, the endogenous allele without targeting event would yield a 1.4-kb band. Lack of such an endogenous band would suggest that both alleles were successfully targeted. (C) Selected clones based on PCR results were confirmed by southern blotting. Parental iPSC (iAAT2) was used as a control. After BamHI digestion of genomic DNA, the endogenous allele without targeting produces a 6.2-kD band. A 4.9-kD band indicates targeting by the HR donor. (D) Sequence analysis showing correction of the Z mutation in AAT patient-specific iPSCs after homologous recombination and piggyBac transposase-mediated seamless excision of integrated donor vector. Z mutation and TALEN recognition sequences are shown. Althoughe the iPSCs derived from the AAT patient showed the G>A mutation (iAAT3), Sanger sequencing of the clone (iAAT3-2) after HR-based gene correction showed a wild-type sequence.

Puromycin-resistant human iPSC colonies obtained after conucleofection of TALEN expression vectors and the targeting vector were screened for targeted events by PCR analyses (Fig. 3B) and verified by southern blotting (Fig. 3C). AAT-iPSC lines derived from 2 different patients were used in this study and both experiments demonstrated high efficiencies of gene targeting (Table 2). Strikingly, among 66 puromycin-resistant iPSC clones that had been expanded and analyzed, all showed the targeted integration of the donor vector based on PCR results (Fig. 3B; Table 2). In addition, 25%-33% of these clones showed the lack of an endogenous allele, suggesting the result of simultaneous targeting of both alleles (Table 2). Six of six candidate clones were confirmed for biallelic gene targeting by southern blotting analysis (Fig. 3C; Table 2).

Table 2. Summary of TALEN-Mediated Homologous Recombination in Two Independent AAT-Deficiency Patient-Specific iPSC Lines (iAAT2 and iAAT3)
Patient iPSC Lines With Z MutationPuro-Resistant Clones ExpandedPositive for Targeted Integration (No. Positive/No. Analyzed)Simultaneously Targeted in Both Alleles (No. Positive/No. Analyzed)
PCRSouthern BlottingPCR*Southern Blotting
  • *

    Numbers are the clones that are positive for targeted integration and negative for endogenous allele.


To achieve a clean gene correction at the AAT locus, we removed the piggyBac-flanked drug-selection cassette from two of the homozygously targeted iPSC clones (iAAT3-2 and iAAT2-33) by transient transfection of a piggyBac transposase-expressing vector,24 followed by drug (fialuridine) selection. The genotype of the resulting colonies was analyzed by PCR (not shown) and DNA sequencing (Fig. 3D). Sequence analyses of selected clones demonstrated that the Z mutation was corrected on both alleles (Fig. 3D).

Functional Characterization of Gene-Corrected iPSCs.

To confirm that the genetic correction of AAT iPSCs resulted in phenotypic correction, these iPSC clones were differentiated into multistage hepatic cells. The corrected iPSCs could efficiently differentiate to MH-like cells (Fig. 4A-C), and there were no significant changes in growth pattern or differentiation kinetics after the gene-modification process. Gene-corrected iPSC clones were able to differentiate into late-stage hepatic cells expressing mature hepatocyte markers, such as cytokeratin 18 (CK18) and albumin (ALB) (Fig. 4A,B). These mature-stage hepatocyte-like cells derived from gene-corrected iPSCs also exhibited metabolic capabilities, as measured by the activities of four major CYP enzymes (CYP3A4, CYP1A2, CYP2C19, and CYP2D6; Fig. 4C), indicating the in vitro functionality of these cells.

Figure 4.

Functional restoration of gene-corrected iPSCs. (A and B) Hepatic differentiation of gene-corrected iPSCs. Gene-corrected iPSC clones were able to differentiate into mature hepatic cells expressing CK18 (A) and ALB (B). Representative images of a gene-corrected clone (iAAT3-6)-derived mature hepatic cells are shown. (×100) (C) These mature-stage hepatic cells derived from gene-corrected iPSCs also exhibit comparable in vitro functional capabilities to healthy control iPSCs, as measured by the activities of four major CYP enzymes (CYP1A2, CYP3A4, CYP2D6, and CYP2C19). (D and E) PASD staining of MH-like cells derived from a patient iPSC line (iAAT3; D) and the gene-corrected iPSC clone (iAAT3-2; E). (×100) (F) Levels of AAT detected within hepatocyte-like cells derived from control-iPSC (iH71), patient-iPSC (iAAT3), and gene-corrected iPSCs (iAAT3-2, red-underlined). Dotted underlines are the effects of the final drug hits in the screening condition (i.e., 5 μM for 4 days) on hepatocyte-like cells derived from the same patient iPSCs.

Importantly, as predicted, the mutant AAT accumulation was no longer detectable in the MH-like cells derived from gene-corrected iPSCs (Fig. 4D,E). The numerous PASD-positive inclusion bodies/globules were observed within hepatocyte-like cells derived from AAT patient iPSCs, whereas these were not detected within hepatocyte-like cells derived from gene-corrected iPSCs (Fig. 4D,E), indicating restored cellular function after gene correction.

In addition, we measured intracellular AAT levels in MH-like cells derived from gene-corrected iPSCs (Fig. 4E) using the same IF-based AAT assay used for the drug-screening process. The AAT level detected within hepatocyte-like cells derived from gene-corrected iPSCs was as low as that of control (healthy donor derived) iPSCs and also comparable to some of the drug-treated (without gene correction) cells, further confirming the functional correction of gene-corrected iPSCs (Fig. 4F).

Therefore, both approaches employed in this study (i.e., gene correction and pharmacological treatment) demonstrated a significant reduction of AAT accumulation within AAT-deficiency patient iPSC-derived hepatocyte-like cells.


Using our patient-specific iPSCs, we screened the clinical-ready drug library (the JHDL) and identified multiple validated hits for novel treatment of AAT deficiency (Table 1), demonstrating the feasibility of iPSC-based drug screening (Figs. 1 and 2). These findings have great implications for developing similar drug-screening platforms for other diseases. Specifically, this proof-of-principle study with AAT-deficiency iPSCs will provide a foundation for iPSC-based preclinical drug discovery and development of novel therapeutics not only for its associated diseases, such as liver cirrhosis and cancer, but also for other complex diseases, such as neurodegenerative disorders caused by pathologic accumulation of misfolded proteins.43

Interestingly, three drugs (Li, CBZ, and VPA) among the final five hits were previously implicated as enhancers of autophagy—a physiological process involved in the clearance of aggregate-prone cytosolic proteins.40, 44 In the case of Li, we found out, after the blind screening, that there were multiple different forms of lithium (i.e., Li-Br, Li-OH, and Li-Cl) within the drug library and all were detected as hits. In addition, one of the compounds, which significantly increased the AAT level (Fig. 2A) (i.e., bovinocidin or 3-nitropropionic acid), has been previously shown to cause brain lesions similar to those of Huntington's disease (HD), which is also caused by protein misfolding.45 Together, three results support that our disease-specific iPSC-based assay is suitable for drug screening as well as further pathogenesis research, and that our findings are not the result of bias or chance. Our screening results, based upon AAT-deficiency patient iPSCs, also indirectly suggest mammalian target of rapamycin (mTOR)-independent autophagy as a potential action mechanism of these drugs, because rapamycin, which inhibits mTOR and a negative regulator of autophagy, did not alter AAT levels in our screening assay system. In addition, we did not observe significant effects of glycogen synthase kinase 3 beta (GSK-3β inhibitors, such as CHIR99021 and histone deacetylase (HDAC), or inhibitors such as sodium phenylbutyrate and sodium butyrate (not shown). Therefore, the effects of Li (a known GSK-3β inhibitor) and VPA (a known HDAC inhibitor) cannot be accounted for by GSK-3β or HDAC inhibition either. It has been recently reported that another HDAC inhibitor, suberoylanilide hydroxamic acid, mediated the correction of AAT deficiency, in part, through HDAC7 silencing46; therefore, the contribution of HDAC inhibition in VPA's effects may require further investigation. Together, our data (Fig. 2 and Supporting Figs. 5 and 6) suggest that the mechanisms by which the final five drugs influence AAT accumulation within hepatocytes are likely to be associated with mTOR-independent autophagy-mediated degradation of folded AAT proteins, rather than decreasing synthesis of AAT. However, we cannot rule out the possibility of other mechanisms, including unconventional autophagy/proteasomal degradation. Thus, the iPSC-based drug-screening and discovery strategy outlined in this report may be applicable to various protein misfolding disorders, including Alzheimer's disease, Parkinson's disease, HD, and amyotrophic lateral sclerosis, in addition to AAT deficiency.43

Importantly, it has been recently shown that the autophagy-enhancing drug, CBZ, decreased the hepatic load of mutant AAT accumulation and hepatic fibrosis in a mouse model of AAT-deficiency–associated liver disease.47 This in vivo finding is consistent with our drug-screening result based upon the human iPSC model of the disease. Together, these results provide a strong basis for testing autophagy enhancers for therapeutic use. Given that most of our drug candidates are already FDA approved and have extensive clinical safety profiles (we also confirmed that these drugs do not influence functionality or viability of hepatocyte-like cells derived from patient and control iPSCs; Supporting Figs. 7-9), there will be no need for further safety tests, which are a main impediment in moving a “hit” to a clinical drug. However, considering their existing use for these drugs and nontoxic therapeutic ranges, it may be necessary to readjust the therapeutic range of certain drugs, including Li, before their new applications (i.e., treating or preventing AAT-deficiency–associated liver disease). The new applications should also avoid unwanted drug interactions, including mutual antagonism, between these drugs (Supporting Fig. 10).

Efficient gene targeting is essential for future iPSC-based gene and cell therapy. Toward this goal, technologies such as ZFN-mediated enhancement of homologous recombination rates in iPSCs have been developed, including gene correction at the AAT locus.24, 25, 27-29, 48 Although it can be highly efficient, the broad application of ZFNs has been limited because of the highly specialized knowledge and tools required for designing functional ZFNs, in addition to high cost. In comparison, the TALEN design has been much more flexible and less costly. Although it is still in the early developmental stage, this technology has shown great potential for many applications, including gene targeting in human stem cells.30-34, 49, 50 Our study, with multiple patient-specific iPSC lines, demonstrates that TALEN-mediated targeting of disease-causing mutations can be a broadly applicable approach to generate isogenic and disease-free sources for cell-replacement therapy. Our results also demonstrated that the TALEN we used in this study can achieve comparable or higher gene-targeting efficiencies (100% efficiency with 25%-33% biallelic targeting) than that observed with ZFNs (54% efficiency with 4% of biallelic targeting) using the exact same targeting vector.24 Because of the different spacer requirements for ZFN- and TALEN-binding sequences, it is not feasible to design the TALEN pairs that recognize the exact same sequences as ZFNs; therefore, a precise comparison of targeting efficiency may not be practical. Nevertheless, our TALENs have significant overlap with the reported ZFNs in their recognition sequences, and the targeting results using the same donor vector suggest that TALEN can achieve a similar targeting efficiency as ZFNs. The results from a reporter assay (Supporting Fig. 11) further support TALEN as an efficient, robust, and economic alternative to ZFN technology. Importantly, our data demonstrate a high efficiency of biallelic gene correction using TALEN, which is fast and cost-effective. Therefore, this approach may be highly compatible with the large-scale production of corrected patient-specific iPSCs for many other monogenic disorders. In addition to the application for gene therapy, it will be widely useful for basic gene-targeting applications, such as creating ideal (i.e., isogenic) controls for iPSC-based disease modeling.

In summary, with emerging new tools and technologies, including patient-specific iPSCs, a clinical-ready drug library, and TALEN, we demonstrated proof of principles for the feasibility of iPSC-based large-scale drug screening and highly efficient gene correction. Integration of patient-specific iPSC-based screening in early stages of drug development will help to more accurately predict drug effects in humans, thereby significantly shortening the timeline and reducing the costs associated with clinical trials and high failure rates. Although many basic or preclinical applications can immediately benefit from our gene-targeting study, gene therapy still warrants further extensive safety studies before translation into the clinic. Nonetheless, our findings have great implications for developing iPSC-based novel therapeutics for the treatment or prevention of currently incurable diseases, including AAT-deficiency–associated liver diseases and other complex disorders, which would benefit from drug screening or gene targeting.